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Release the B7.1! Anti-PD-L1 breaks PD-L1’s grip

April 1, 2020

Blocking the PD-1/PD-L1 axis using anti-PD-L1 antibodies is a commonly employed immunotherapeutic strategy to prevent PD-1-mediated inhibition of T cells by tumor cells. Suspecting that the mechanism of action of PD-L1 blockade might be more complex, however, Mayoux et al. investigated the role of PD-L1-expressing dendritic cells (DCs) in PD-L1 blockade. The results were recently published in Science Translational Medicine.

When a DC primes a T cell, the generally understood mechanism is that B7.1 on dendritic cells engages with the positive costimulator CD28 on T cells, encouraging T cell activation. If the negative costimulator PD-1 is also engaged on T cells, then T cell activation could be inhibited. Mayoux et al. investigated the expression of PD-L1, PD-1, and the CD28 stimulatory ligand B7.1 (aka CD80) on various DC subsets from patients with lung cancer and found that both tumor-associated and peripheral DCs expressed PD-L1. They also observed that tumor-associated but not peripheral cross-presenting DCs (CD141+ and CD1c+) expressed B7.1. Focusing on a double-positive (PD-L1+ B7.1+) population in cross-presenting DCs from fresh renal cell carcinoma samples, and cognizant that PD-L1 and B7.1 have been known to interact in cis, the researchers used an antibody binding assay to quantify surface expression and observed that PD-L1 was 20-fold more abundant than B7.1 on both CD141+ and CD1c+ conventional dendritic cells.

Based on this evidence, Mayoux et al. hypothesized that the abundant PD-L1 could sequester the less abundant B7.1 in cis on DCs, leaving excess PD-L1 available for binding. To test this, they used monocyte-derived DCs from healthy donors in an in vitro system to develop LPS-matured DCs and observed expression patterns of PD-L1 and B7.1 that were similar to those observed in tumors. In a confocal imaging analysis, when the DCs were observed forming synapses with T cells, PD-1 and CD28 colocalized more often than PD-1 and PD-L1, consistent with recent findings that PD-1 signaling may be involved in the dephosphorylation of CD28 at the immunological synapse. Interestingly, B7.1 was barely observed colocalizing with CD28 at all, instead interacting to a much higher degree with PD-L1 in cis, likely due to its higher binding affinity.

To test whether blocking PD-L1 might release B7.1 from sequestration and make it available for interaction with CD28, the researchers modeled the physical interactions between B7.1/PD-L1 and B7.1/CD28. First, they showed that anti-PD-L1 binds to PD-L1-expressing cells with a binding affinity 700-fold higher than the binding affinity between PD-L1 and B7.1, suggesting that the antibody would readily disrupt PD-L1/B7-1 binding. Next, using cells engineered to mimic the expression of B7.1 and PD-L1 on DCs, and CD28-Fc to mimic T cells, the researchers observed an increase in B7.1/CD28 interactions when anti-PD-L1 was present. These results were confirmed in a primary cell-cell system, which also showed that pre-incubating mature DCs with anti-PD-L1 enabled stronger interactions between B7.1 on DCs and CD28 on T cells. Together this evidence indicated that anti-PD-L1 can disrupt the interatctions between PD-L1 and B7.1 in cis, freeing up B7.1, enhancing B7.1 binding to CD28, and activating T cells.

To measure the functional consequences of anti-PD-L1 disrupting the binding between PD-L1 and B7.1 in cis on DCs, Mayoux et al. evaluated CD28 downstream signaling in Jurkat reporter cells. To better isolate the specific contribution of blocking the interaction between PD-L1 and B7.1 in cis, Jurkat cells were first incubated with anti-PD-1 to block PD-1/PD-L1 binding in trans, which increased CD28 signaling by 32%. The cells were then incubated with anti-PD-L1, which further enhanced CD28 signaling by 20%, for an overall increase of 52% in CD28 binding. When the researchers repeated the experiment using a PD-L1-targeting antibody that does not substantially block PD-L1/B7.1 interactions, the increase in CD28 signal transduction was significantly lower.

Next, Mayoux et al. studied the consequences of pretreating activated human DCs with PD-L1 blockade and found that it directly enhanced their ability to stimulate the proliferation of allogeneic T cells. To study whether this effect could generate de novo immune responses, mouse splenic CD11c+ DCs were cocultured with DQ-OVA (cross-presented as the SIINFEKL peptide) and treated with anti-PD-L1. In coculture with naive T cells from OT-I mice, the DCs pretreated with anti-PD-L1 induced more T cell proliferation; increased T-bet and Eomes in T cells, suggesting enhanced T cell effector potential; and enhanced production of granzyme B in T cells in response to target cells. Similar results observed using SIINFEKL peptide-pulsed DCs pretreated with anti-PD-L1.

To test the extent to which tumor-associated DCs contribute to responses to PD-L1 blockade in a clinical setting, Mayoux et al. established a DC gene signature based on RNAseq data and quantification of expression of key genes that defined DC subsets. In patients with renal cell carcinoma treated with atezolizumab (anti-PD-L1), patients with a high pretreatment DC signature showed better overall survival. In patients with non-small cell lung cancer treated with either anti-PD-L1 or docetaxel, a high DC signature correlated with increased overall survival only in patients in the anti-PD-L1 treatment group. Further, the survival benefit was only observed when PD-L1 was expressed on immune cells, refining the correlation between a high DC signature and increased overall survival, and suggesting that these factors could be considered when making decisions regarding treatment options.

Overall, Mayoux et al. show that the activation of T cells by antigen-presenting cells involves a complex network of interactions between PD-1, PD-L1, B7.1, CD28, and possibly CTLA-4 (not yet investigated). They also show that intratumoral DCs expressing PD-L1 and B7.1 likely play a significant role in the outcome of PD-L1 blockade. In addition to blocking interactions between PD-L1 and PD-1, anti-PD-L1 antibodies also prevent interactions between PD-L1 and B7.1 in cis on dendritic cells, freeing up B7.1 to interact more readily with CD28 on T cells and prime T cell activation and antitumor responses – a mechanism not available to anti-PD-1 antibodies. Importantly, the presence of a DC signature could help guide treatment decisions in the clinical setting.

by Lauren Hitchings


Mayoux M., Roller A., Pulko V., Sammicheli S., Chen S., Sum E., Jost C., Fransen M.F., Buser R.B., Kowanetz M., Rommel K., Matos I., Colombetti S., Belousov A., Karanikas V., Ossendorp F., Hegde P.S., Chen D.S., Umana P., Perro M., Klein C., Xu W. Dendritic cells dictate responses to PD-L1 blockade cancer immunotherapy. Sci Transl Med. 2020 Mar 11.

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